據估計，全世界每年約有 10 億例人類真菌感染病例和 150 萬例死亡。此外，真菌感染也會嚴重影響動植物。例如，真菌病會導致主食大量流失，從而對經濟和生計造成嚴重影響。儘管某些類型的抗真菌藥物可用於治療真菌感染，但選擇的數量不足。因此，迫切需要了解抗真菌藥物的作用機制以及真菌細胞如何抵抗它們。先前使用粉色麵包黴菌Neurospora crassa作為模型真菌的研究表明，czt-1(cell death-activated zinc cluster transcription factor ;一種調節 ATP 結合盒轉運蛋白表達的轉錄因子）與對細胞死亡誘導劑星形孢菌素 (STS) 的抗性之間存在關聯。在野生種群中，czt-1 的遺傳變異與另一個轉錄因子基因 tah-3 的表達有關，這也可能導致耐藥性。 CZT-1和TAH-3之間的分子關係尚不清楚。當我測試 Δczt-1 和 Δtah-3 敲除突變體對一組臨床相關藥物的敏感性時，我觀察到 tah-3 的缺失導致對卡泊芬淨、氟康唑、伊曲康唑和伏立康唑的敏感性增加。我還創建了一個同時缺乏 czt-1 和 tah-3 的菌株，並檢查了雙突變體與其親本單突變體相比是否具有不同的藥物敏感性，但 Δczt-1Δtah-3 細胞對兩者中最敏感的細胞表現出相同的表型父母。這不允許我對這兩個基因之間的遺傳相互作用做出結論。另一方面，我還研究了 czt-1 (L680F) 中的一個點突變，該突變以前曾被認為會導致耐藥性增加。為了更準確地研究 L680 殘基的重要性，我構建了 czt-1 的野生型和 L680F 等位基因的 GFP 標記版本，並通過共軛焦顯微鏡顯示 CZT-1L680F 在細胞中的含量更高，這表明L>F 突變可能導致 CZT-1 表達的正反饋。此外，在暴露於 STS 的細胞或過表達 TAH-3 的細胞中，CZT-1wild type斑點的數量顯著增加。通過分析預測的 CZT-1 三維結構，我觀察到 L680 殘基位於長 α-螺旋的最末端，這可能與細胞環境中的元素（例如其他蛋白質或隔室（例如細胞膜））相互作用.此外，我使用轉基因技術創建了新的工具，這些工具在未來將促進對 CZT-1 和 TAH-3 作用的功能研究。具體來說，我構建了兩個 TAH-3-mCherry 構建體和兩個額外的 CZT-1 突變等位基因（CZT-1L680R-GFP 和 CZT-1L680D-GFP）。總之，我的工作有助於進一步了解 CZT-1 和 TAH-3 在抗真菌藥物反應過程中的作用，儘管需要更多的研究來全面表徵它們在真菌細胞中的功能。

關鍵詞: 粉色麵包黴菌, czt-1, tah-3, 真菌抗藥性

It is estimated that there are approximately 1 billion cases and 1.5 million deaths from fungal infections in humans, worldwide, each year. In addition, fungal infections can also severely affect plants and animals. For example, fungal diseases can lead to massive losses of staple foods, which can have harsh impacts on economies and livelihoods. Although some types of antifungal drugs are available to treat fungal infections, the number of options is insufficient. Therefore, it has become urgent to understand the mechanisms of action of antifungal drugs and how fungal cells resist to them. Previous studies using Neurospora crassa as a model fungus demonstrated an association between czt-1, a transcription factor that regulates the expression of ATP-binding cassette transporters, and resistance to the cell death inducer staurosporine (STS). In wild populations, genetic variants of czt-1 are associated with the expression of another transcription factor gene, tah-3, which may also contribute to drug resistance. The molecular relationship between CZT-1 and TAH-3 is not clear. When I tested the susceptibility of Δczt-1 and Δtah-3 knockout mutants to a panel of clinically relevant drugs, I observed that loss of tah-3 resulted in increased sensitivity to caspofungin, fluconazole, itraconazole and voriconazole. I also created a strain that lacks both czt-1 and tah-3 and examined whether the double mutant had different drug susceptibility compared to its parental single mutants, but Δczt-1Δtah-3 cells showed an identical phenotype to the most sensitive of the two parents. This did not allow me to make conclusions about genetic interactions between these two genes. On the other hand, I also studied a point mutation in czt-1 (L680F), which had previously been suggested to cause increased drug resistance. To more precisely investigate the importance of the L680 residue, I constructed GFP-tagged versions of wild type and L680F alleles of czt-1 and showed, by confocal microscopy, that CZT-1L680F was present in higher amounts in the cells, suggesting that a potential constitutive activation triggered by the L>F mutation could lead to positive feedback for CZT-1 expression. Furthermore, the number of CZT-1wild type puncta was strongly increased in cells exposed to STS or in cells overexpressing TAH-3. By analyzing a predicted tridimensional structure of CZT-1, I observed that the L680 residue is at the very end of a long α-helix, which may be involved in interactions with elements of the cellular environment such as other proteins or compartments such as membranes. In addition, I used molecular cloning to create new tools that in the future will facilitate functional studies on the role of CZT-1 and TAH-3. Specifically, I built two TAH-3-mCherry constructs and two additional CZT-1 mutant alleles (CZT-1L680R-GFP and CZT-1L680D-GFP). In summary, my work has helped to further understand the role of CZT-1 and TAH-3 during the response to antifungal drugs, although more studies are needed to comprehensively characterize their functions in fungal cells.

Keyword: Neurospora crassa, czt-1, tah-3, antifungal drug resisitance

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